Correlation between choline kinase alpha expression and 11C-choline accumulation in breast cancer using positron emission tomography/computed tomography: a retrospective study

Choline kinase (CK) is reportedly overexpressed in various malignancies. Among its isoforms, CKα overexpression is presumably related to oncogenic change. Choline positron emission tomography (PET) is reportedly useful for detecting and evaluating therapy outcomes in malignancies. In this study, we investigated the correlation between CKα expression and 11C-choline accumulation in breast cancer cells. We also compared the CKα expression level with other pathological findings for investigating tumour activity. Fifty-six patients with breast cancer (mean age: 51 years) who underwent their first medical examination between May 2007 and December 2008 were enrolled. All the patients underwent 11C-choline PET/computed tomography imaging prior to surgery. The maximum standardised uptake value was recorded for evaluating 11C-choline accumulation. The intensity of CKα expression was classified using immunostaining. A significant correlation was observed between CKα expression and 11C-choline accumulation (P < 0.0001). A comparison of breast cancer mortality demonstrated that strong CKα expression was associated with a shorter survival time (P < 0.0001). 11C-choline accumulation was also negatively correlated with survival time (P < 0.0001). Tumours with strong CKα expression are reportedly highly active in breast cancer. A correlation was observed between CKα expression and 11C-choline accumulation, suggesting their role as prognostic indicators of breast cancer.


Patients
In this study, 56 patients (mean age, 51 years; range 24-71 years) with breast cancer were enrolled.The patients underwent their first medical examinations between May 2007 and December 2008.All the patients underwent 11 C-choline PET/CT.The diagnosis of breast cancer was confirmed by biopsy at least 1 month prior to the imaging studies.Patients with a performance status of 2 or more and those with multiple simultaneous malignancies were excluded.Hormone therapy was administered upon completion of all the imaging studies, and patients who had already received hormone therapy were excluded.Patients who underwent surgery after the imaging studies were included.Written informed consent was obtained from all the patients.This study was approved by the Institutional Review Board of the National Cancer Centre Hospital, Tokyo, Japan.This study complied with the guidelines of the Health Insurance Portability and Accountability Act.

Phantom study
Imaging was performed using a whole-body PET/CT scanner (Aquiduo PCA-7000B; Toshiba Medical Systems, Tochigi, Japan).Prior to the study, a phantom study was conducted at two facilities to ensure imaging quality 17 .A NEMA phantom (NU 2-2001) was used for the study.The background radioactivity concentration was set to 2.6 ± 0.2 kBq/mL, which is close to the imaging conditions for clinical use.The radioactivity concentration of the hot portion was set four times greater than that of the background.Data were collected for 2-5 and 30 min in the dynamic and static acquisition modes, respectively.The data were visually assessed and used to evaluate the phantom noise equivalent count (NEC phantom ), percentage contrast of the hot portion (Q H10mm ), and percentage background variability (N 10 mm ).The reference values for the physical indices were as follows: NEC phantom > 10.4 (counts), N 10mm < 6.2%, and Q H10mm /N 10mm > 1.9%.After the phantom study, the imaging conditions were set as follows: data acquisition, 180 s for one bed; field-of-view, 500 mm; iterations, 4; subsets, 14; matrix size, 128 × 128; filter, Gaussian 8 mm in full width at half maximum; and reconstruction, ordered subset expectation maximisation.

Data acquisition
The synthesis of 11 C-choline was based on the study by Hara et al. 18 .The patients fasted for at least 6 h before the imaging examinations.After urination, the patients were placed in a supine position with both arms raised.First, plain CT imaging was performed from the top of the head to the mid-thigh under free-breathing, at 120 KVp using an autoexposure control system (beam pitch, 0.875 or 1; and 1.5 or 2 mm × 16-row mode).Within 5 min of the intravenous injection of 11 C-choline (average, 475.5 MBq; range, 457-491 MBq), PET imaging was performed for the patient's head to the mid-thigh.

Image interpretation
CT, PET, and composite images were reviewed using proprietary software (Vox-base SP1000 workstation; J-MAC Systems, Sapporo, Japan).Two independent evaluators performed the visual and quantitative assessments.The findings were documented based on a consensus.The region of interest (ROI) was set as the contour area of increased uptake.If the uptake was heterogeneous, the ROI was set to cover the entire area.The standardised uptake value (SUV) was quantitatively recorded.The SUV max was determined as the maximum accumulation within the ROI.Time-decay correction was not performed.

Pathologic analysis
All patients underwent surgery.Surgical materials were fixed in 10% formalin and embedded in paraffin.Slices of 4 μm were prepared perpendicular to the long axis of the breast.Histological and nuclear grades were evaluated using the Elston-Ellis scoring system 19 .Oestrogen and progesterone receptor (ER and PgR, respectively) expression was evaluated using the H-scoring system described by McCarty et al. 20 .Human epidermal growth factor-2 (HER-2) immunostaining was performed using a 4B5 primary antibody.CKα immunostaining was performed using ab235938 antibody (ATLRAS 1-100; Atlas Antibodies AB; Sweden), and its expression was evaluated based on three grades from 0 to 2 (0, low intensity; 1, moderate intensity; and 2, high intensity).The following items were compared with CK α expression of invasive component, lymphatic invasion, histologic grade, nuclear grade, nuclear atypia, mitosis, extensive intraductal components, fat invasion, cutaneous invasion, muscular invasion, HER-2/neu, ER, and PgR.

Correlation between CKα expression and 11 C-choline accumulation using PET
All the primary tumours were evaluated using 11 C-choline PET/CT (Fig. 1).The mean SUV max of all the cases was 3.2 (SD ± 1.8; range 0.95-8.3).After immunostaining for CKα, 18 (32%), 16 (29%), and 22 (39%) tumours were categorised as grades 0, 1, and 2, respectively.The correlation between the strength of CKα expression and SUV max of 11 C-choline PET is shown in Tables 2 and 3.The box plots are shown in Fig. 2a and b.A significant correlation was observed between these two variables (P < 0.0001).The correlation persisted upon comparing the weak (CKα expression graded as 0 or 1) and strong expression (CKα expression graded as 2) groups.

Correlation between CKα expression and 11 C-choline accumulation and mortality
The patients' medical records were evaluated until July 2022.During this period, 19 deaths owing to breast cancer were confirmed.Among these 19 cases, the mean survival time was 101 months (SD ± 36; range 18.7-176).The correlation between the strength of CKα expression and breast cancer mortality is shown in Tables 4 and 5.The Kaplan-Meier curves are shown in Fig. 3a and b.A significant difference was observed in the survival time based on the expression intensity of CKα (P < 0.0001).Survival time also significantly differed between the high-SUV (SUV max > 3) and low-SUV (SUV max ≤ 3) groups (P < 0.0001) (Table 6 and Fig. 3c).www.nature.com/scientificreports/

Correlation between the pathological findings and CKα expression
The correlations between the pathological findings and CKα expression strength are listed in Tables 7 and 8. Significant correlations were observed between CKα expression and the invasive components, mitosis, extensive intraductal components, fat invasion, ER, and PgR.Comparison of the weak and strong expression groups revealed significant differences in the invasive components, mitosis, extensive intraductal components, fat invasion, and PgR.No correlation was observed with ER.

Discussion
There have been several reports regarding 11 C-choline accumulation in breast cancer.However, the relationship between the mechanism of 11 C-choline accumulation and the pathological background had never been investigated.The present study demonstrated a correlation between CKα expression and 11 C-choline accumulation using PET.Associations between 18 F-fludeoxyglucose (FDG) and 11 C-choline accumulation and the histological findings in breast cancer were demonstrated in a previous study 16 .Thus, mitosis correlated with 11 C-choline accumulation.No similar relationship was observed between mitosis and 18 F-FDG accumulation.As previously mentioned, CK overexpression promotes mitogenic progression 2 .This can be explained from a biochemical perspective.Similar to that in breast cancer, a strong correlation between 11 C-choline accumulation and CKα expression has been reported in prostate cancer 15 .CKα was suggested to be involved in choline metabolism in these malignancies.However, in gliomas, 18 F-choline accumulation reportedly did not correlate with CKα expression 21 .Increased expression of mRNA and protein of CK in lung cancer has been reported; however, no correlation was observed between 11 C-choline accumulation and these factors 22 .The small number of cases is one of the limitations of both studies.However, CKβ or other synthetic pathways may be predominant according to the type of malignancy.
We investigated the correlation between the strength of CKα expression and the pathological findings of breast cancer.As shown above, significant correlations were observed between CKα expression and the invasive components, mitosis, extensive intraductal components, and fat invasion.These findings suggest a relationship between CKα and breast cancer activity.According to a previous study 4 , CK enzymatic activity and overexpression, as measured using western blotting, correlate with the histologic grade.In the current study, CKα expression was examined using immunostaining, and no correlation was observed with the histological grade.Differences in the measurement methods could be attributed to this finding.The same study also demonstrated correlations among CK activity, overexpression, and ER deficiency.Although no association with ER was observed in the current study, an association between CKα expression and PgR deficiency has been suggested.This difference in hormone receptor expression may also arise from different measurement methods.We suggest that breast cancers with strong CKα expression tend to be hormone receptor-negative.
There are few reports comparing CKα expression and pathological findings to examine tumour activity in other malignancies.In the report on gliomas mentioned above, only one sample was strongly positive for CKα, and no significant relationship with tumour grade was observed 21 .According to a previous report on hepatocellular carcinoma, there was no relationship between CKα expression and tumour grade.However, CKα expression correlated with the cancer stage 23 .It is unclear whether the correlation between CKα expression and tumour activity is common among malignancies.Thus, we need further research.
In our present study, we found a significant correlation between CKα expression and survival time in breast cancer, suggesting that strong CKα expression was associated with a bad prognosis.A similar relationship between CKα and short survival time has been reported in hepatocellular carcinoma and non-small-cell lung cancer 23,24 .A correlation between survival time and the SUV max of 11 C-choline was also observed in the present study. 11C-choline accumulation has been suggested to correlate with tumour aggressiveness 14 , and our results are consistent with this report.Both CKα expression and 11 C-choline accumulation could be potential prognostic factors for breast cancer.18 F-FDG is the most widely used among PET formulations.A previous study targeting breast cancer reported that 18 F-FDG accumulation had a significant correlation with both recurrence and mortality 25 .According to a meta-analysis, patients with high SUV max of 18 F-FDG in the primary lesion had a shorter event-free survival time.However, 18 F-FDG accumulation has no significant correlation with overall survival 26 .A previous study targeting patients with bone metastases also reported that high 18 F-FDG accumulation was associated with skeletal-related events and progression, but not with overall survival 27 . 11C-choline, which we used in the present study, might

Figure 1 .
Figure 1.Imaging and pathologic findings of a 50-year-old woman.(a) 11 C-choline positron emission tomography/computed tomography image.A focal accumulation was observed in the primary tumour (maximum standardised uptake value [SUV max ] = 8.3).(b) Result of haematoxylin-eosin staining.A diagnosis of invasive ductal carcinoma was made.Both histological and nuclear grades were classified as 3. (c) Result of choline kinase alpha (CKα) immunostaining.High-intensity staining was observed.

Table 1 .
Patient demographics.Data are presented as mean ± standard deviation (range) or the number of cases.The data in parentheses are presented as percentages.NST No special type.

Table 2 .
Correlation between CKα expression and 11 C-choline accumulation using PET.SUV ma , Maximum standardized uptake value; PET Positron emission tomography; CKα Choline kinase alpha.

Table 3 .
Correlation between CKα expression and 11 C-choline accumulation using PET (CKα 0-1 vs. 2).Data are presented as mean ± standard deviation (range) or the number of cases.The data in parentheses are presented as percentages.SUV max Maximum standardized uptake value; PET Positron emission tomography; CKα Choline kinase alpha.

Table 6 .
11rrelation between11C-choline accumulation and mortality.Data are presented as mean ± standard deviation or the number of cases.The data in parentheses are presented as percentages.SUV max Maximum standardized uptake value.

Table 8 .
Correlation between the pathological findings and CKα expression (CKα 0-1 vs. 2).Data are presented as the number of cases.The data in parentheses are presented as percentages.HER-2 Human epidermal growth factor-2; CKα Choline kinase alpha.